ES2887404T3 - Compositions for introducing nucleic acid into cells - Google Patents

Abstract

A random copolymer comprising a plurality of repeat units (a) independently selected from repeat units of the following formulas (a1) and (a2): **(See formula)** and a plurality of repeat units (b) selected independently of repetition units from the following formulas (b1) to (b4): **(See formula)** in which the molar ratio of the sum of the repetition units (a) with respect to the sum of the repeating units (b) is within the range of 0.7/1.0 to 1.0/0.7, and wherein one or more of the nitrogen atoms of repeating units (a) and/or or (b) contained in the copolymer can be protonated to provide a cationic copolymer.

Description

DESCRIPTION

Compositions for introducing nucleic acid into cells

The present invention relates to polymers comprising characteristic alkyleneamine repeat units that are useful as vehicles for transfecting a cell with a nucleic acid, in particular RNA. The present invention further relates to a composition comprising at least one nucleic acid and a polymer comprising such alkyleneamine repeat units and a method of transfecting a cell using said composition. Furthermore, the present invention relates to pharmaceutical compositions and their uses.

The viability of nucleic acid therapies ultimately depends on the availability of efficient methods to deliver nucleic acids to cells.

In nucleic acid delivery in general, the use of naked nucleic acids is adequate and in some cases sufficient to transfect cells (Wolff et al. 1990, Science, 247, 1465-1468). However, in most envisioned practical applications, it is advantageous or even necessary to formulate the nucleic acid with at least one second agent that protects the nucleic acid from degradation during delivery and/or facilitates distribution to and in a target tissue and /or facilitate cellular uptake and allow adequate intracellular processing. Such nucleic acid delivery formulations are referred to as vectors in the scientific literature. A wide variety of compounds for targeting nucleic acids, so-called transfection reagents, have been previously described. These compounds are usually either polycations or compositions comprising cationic lipids or lipid-like compounds such as lipidoids (US 8,450,298). Complexes of nucleic acids with polycations are called polyplexes, those with cationic lipids are called lipoplexes (Felgner et al. 1997, Hum Gene Ther, 8, 511-512). Complexes comprising both a polycation and lipids have also been described (Li and Huang in "Nonviral Vectors for Gene Therapy", Academic Press 1999, chapter 13, 295-303). Transfection reagents are used to bind and compact nucleic acids to result in primary complexes in the nanometer size range. In salt-containing media, these complexes tend to aggregate, also known as salt-induced aggregation, which may be advantageous for transfection in cell culture or localized delivery in vivo (Ogris et al. 1998, Gene Ther, 5, 1425- 1433; Ogris et al. 2001, AAPS PharmSci, 3, E21). Aggregation can be prevented and complexes of nucleic acids with transfection reagents can be stabilized by surface protection with polymers such as polyethylene glycol. Protection is also used to prevent opsonization of and activation of complement by nucleic acid complexes with transfection reagents (Finsinger et al. 2000, Gene Ther, 7, 1183-1192). The compaction of nucleic acids by transfection reagents not only protects them against degradation by nucleases, but also renders them suitable for cellular uptake by endocytosis. Numerous linear and branched polycations are suitable for binding and compacting nucleic acids including, but not limited to, poly(ethyleneimine), poly(amidoamine) dendrimers, poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), or derivatives. cationic poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), poly(beta-aminoester)s (Akinc et al., 2003, Bioconj Chem 14(5):979-88), peptides or poly(amino acids) natural and synthetic cationics such as poly(lysines), histones, HMG proteins or cationic carbohydrates such as chitosans. In addition to polymers containing primary, secondary, and/or tertiary amines, the aforementioned structures containing guanidyl moieties are an important class of molecules for the purpose of nucleic acid complexation and delivery. Guanidyl-modified polymers as arginine-based frameworks (Yamanouchi et al. 2008, Biomaterials 29(22):3269-77), arginine-modified PAMAM (Son et al. 2013, Bull. Korean Chem. Soc. Vol 34 n. 3) or guadinylated PEI (Lee et al. 2008, Bull. Korean Chem. Soc. 2008, vol. 29, no. 3) have highlighted the efficiency of such systems. Especially in the case of interaction with RNA, the molecular features of the guanidyl moiety exhibit unique binding properties (Calnan et al. 19991, Science 252(5009), 1167-1171). For the generation of such structures, methods reviewed by Katritzky and Rogovoy (Katritzky & Rogovoy 2005, ARKIVOC (iv) 49-87) can be used. Polyplexes are often further modified to contain a cell targeting or intracellular targeting moiety and/or a membrane destabilizing component such as an inactivated virus (Curiel et al. 1991, ProcNatl Acad Sci USA, 88, 8850-8854 ), a viral capsid or a viral protein or peptide (Fender et al. 1997, Nat Biotechnol, 15, 52-56, Zhang et al. 1999, Gene Ther, 6, 171-181) or a membrane disruptive synthetic peptide ( Wagner et al., 1992, Proc Natl Acad Sci USA, 89, 7934-7938, Plank et al., 1994, J Biol Chem, 269, 12918-12924).

Following endocytic uptake, the complexes are sequestered in intracellular vesicles such as endosomes and lysosomes where they are exposed to cellular degradation machinery. Thus, it has been recognized that escape from intracellular vesicles is essential for the efficient delivery of functional nucleic acid, a requirement that also applies for functional viral infection (Wagner et al. 1992, Proc Natl Acad Sci USA, 89 , 7934-7938, Plank et al. 1994, J Biol Chem, 269, 12918-12924). The mechanisms that nature has evolved for viral infectivity have been mimicked to achieve efficient nucleic acid delivery by synthetic vectors. For this purpose, amphiphilic membrane disrupting peptides such as INF, GALA and KALA peptides or melittin and melittin derivatives (Boecle et al. 2006, J. Control Release, 112, 240-248) have been used with great success to complement polycationic transfection reagents with endosomal escape functionality (Plank et al. 1998, Adv Drug Deliv Rev, 34, 21-35). In lipoplexes, such functionality is inherent in the ability of their lipid moieties to fuse with cell membranes (Xu and Szoka 1996, Biochemistry, 35, 5616-5623, Zelphati and Szoka 1996, Proc Natl Acad Sci USA, 93, 11493-11498) . Since the seminal article by Boussif et al. (Boussif et al. 1995, Proc Natl Acad Sci USA, 92, 7297-7301) it is known that endosomal escape functionality of polyplexes can be realized by physicochemical means. When poly(ethyleneimine) (PEI) is used as the polycation to form polyplexes, its buffering capacity at acidic pH is sufficient to trigger endosomal escape. The lumen of endosomes is known to be acidified by a proton pump residing in endosomal membranes (Lafourcade et al. 2008, PLoS One, 3, e2758). This acidification is the trigger for endosomal escape of some viruses such as influenza or adenovirus. The so-called proton sponge theory, supported by experimental evidence, describes the putative mechanistic action of polymers comprising chemical structural features of PEI: A substantial fraction of the amino groups of PEI are unprotonated at neutral (physiological) pH (Ziebarth and Wang 2010, Biomacromolecules, 11, 29-38). By virtue of the protonated and thus positively charged amino groups, PEI-type polymers can bind and compact nucleic acids. Unprotonated amines can be protonated at acidic pH and thus have buffering capacity within endosomes. Endosomal acidification by the proton pump is accompanied by accumulation of chloride ions (Sonawane et al. 2003, J Biol Chem, 278, 44826-44831). In the presence of a buffering molecule such as PEI in the endosomal lumen, the proton pump will transport many more protons into the endosomal lumen, along with accumulation of chloride, as it would in its absence until the natural acidic endosomal pH is reached. The disproportionate accumulation of ions within the endosomes is thought to lead to osmotic destabilization of the vesicles, ultimately leading to vesicle rupture and release of the nucleic acid complex into the cytoplasm.

Based on the proton sponge theory, numerous researchers have captured the structural features of PEI in the creation of novel polymer libraries comprising amines with buffering capacity at acidic pH. In US 7,780,957 and US 7,829,657, Kataoka et al. describe polymers based on a poly(glutamic acid) or poly(aspartic acid) backbone where the carboxylic acid side chains are derivatized with protonatable amine side chains at acidic pH. However, the rich framework space of oligo(alkyleneamines) containing alternating, non-identical alkyleneamine units to serve as transfection-enhancing moieties into polycations has not been explored. In particular, mRNA transfection has not been previously investigated.

In contrast, much of the scientific work by Kataoka et al. has focused on poly{N-[N'-(2-aminoethyl)-2-aminoethyl]aspartamide}. In a publication by Uchida et al. (2011, J Am Chem Soc, 133, 15524-15532), the same group has examined a series of N-substituted polyaspartamides that possess repeating aminoethylene units in the side chains of the general formula -(CH2-CH2-NH) mH. Interestingly, when the authors examined the efficiency of the polymer family in transfecting plasmid DNA, “a distinctive odd/even effect of repeating aminoethylene units in the polymer side chain was observed on efficiencies of endosomal escape and plasmid DNA transfection.” transfection to various cell lines. Polyplexes of polymers with an even number of repeating aminoethylene units (PA-E) achieved an order of magnitude higher transfection efficiency, without marked cytotoxicity, than that of polymers with an odd number of repeating aminoethylene units (PA-E). repetition (PAO). This odd-even effect was in good agreement with the buffering capacity of these polymers, as well as their ability to selectively disrupt membrane integrity at endosomal pH, leading to highly efficient endosomal escape from PA-E polyplexes. Furthermore, it was shown that the formation of a multivalent charged matrix with precise spacing between protonated amino groups in the polymer side chain was essential for the efficient disruption of the endosomal membrane, thus facilitating the transport of the polyplexus into the cytoplasm” (Abstract from Uchida et al., 2011, J Am Chem Soc, 133, 15524-15532). Interestingly, when the same group of researchers compared poly(aspartamide) derivatives bearing 1,2-diaminoethane side chains, [PAsp(DET)] versus analogs bearing 1,3-diaminopropane side chains, [PAsp(DPT )], observed that PAsp(DPT) polyplexes showed a significant drop in plasmid aDn transfection efficiency at high N/P ratios due to progressively increased cytotoxicity with N/P ratio, despite differences Physicochemical differences with respect to [PAsp(DET)] on particle size and C-potential were negligible (Miyata et al. 2008, J Am Chem Soc, 130, 16287-16294). Therefore, based on the odd-even rule, polymers comprising 3 protonatable amino groups and propylene spacer groups would be expected to be inferior to PAsp(DET) and that side chains comprising 1,3-diaminopropane are associated with toxicity problems. Nothing is known about the structure-activity relationships of such polymers for mRNA transfection.

M. Kramer, “Polymeric Nanocarriers with Dendritic Core-Shell Architectures”, Dissertation, Albert-Ludwigs-Universitat Freiburg i.Br., 2004, describes gene transfection using PEI-based dendrimer particles that can be grafted with side chains or end groups of propylenimine.

Geall et al. have described cholesterol-polyamine carbamates with the polyamine moiety having the general formula:

-NH-CH2-( 'CH2 ')„-CH2-NH-CH2-( 'CH2 ' )m-CH2-NH-CH2-( vCH ¿ 2) ,n n-NH ¿ 2, d ^where m = „ 0, „ 1 or 2 „ and d ,ond ,en = 0 or 1 (Geall et al. 1999, FEBS Lett, 459, 337-342). They have examined the pKa values of these substances and their characteristics in calf thymus DNA condensation. They found that the regiochemical distribution of positive charges along cholesterol-polyamine carbamates plays significant roles in the modulation of DNA binding affinity and lipofection efficiency. They found that among the cholesterolpolyamine carbamates examined, spermine which makes up the polyamine moiety, -HN-CH ² -CH ² -CH ² -NH-CH ² -CH ² -CH ² -CH ² -NH-CH ² -CH ² - CH ² -NH ² (propyl/butyl/propyl) produced by far the highest reporter gene expression upon transfection of plasmid DNA encoding beta-galactosidase in cell culture, whereas, for example, -HN-CH ² -CH ² -NH-CH ² -CH ² -CH ² -NH-CH ² -CH ² -NH ² (ethyl/propyl/ethyl) was three to ten times less efficient. Therefore, in view of the teachings of Kataoka et al. (odd-even rule) and the findings of Geall et al., the skilled artisan would rule out the latter structure in the context of nucleic acid delivery.

Wang et al. have described poly(methyl methacrylate)-graft-oligoamines as efficient and poorly cytotoxic transfection reagents for plasmid DNA (Wang et al. 2010, Molecular BioSystems, 6, 256-263). These polymers were obtained by aminolysis of poly(methyl methacrylate) with oligoamines of the general formula 1H ² N-CH ² -CH ² -(NH-CH ² -CH ² ) ^m -NH ² , where m = 1, 2 or 3 The authors found that transfection efficiency increased with increasing amine length.

Oh et al. have described poly(disulfuroamidoamines) that are derived from capped oligoamines having the structure Dde-NH-(CH ² ) ^a -NH-(CH ² ) ^b -NH-(CH ² ) ^a -NH-Dde by copolymerization with N ,N'-cystaminebisacrylamide (Ou et al. 2009, Biomaterials 30, 5804-5814; WO 2010/065660). They examined the combinations a = 2 and b = 2, a = 2 and b = 3, a = 3 and b = 2, a = 3 and b = 3, a = 3 and b = 4 (spermine). Dde is the protecting group of 2-acetyldimedone. After removal of the protecting group, the synthesis produces poly(disulfuroamidoamines) where the internal, originally secondary amines are converted to tertiary amines as part of the polymer backbone and the terminal amines become part of the ethylene or ethylene side chains. pendant propyleneamine. Such polymers have buffering capacity in the pH range relevant to nucleic acid delivery and are useful for transfecting plasmid DNA into cells.

Recently, the utility of a new class of synthetic lipid-like but non-lipid structures, the so-called lipidoids, for in vitro and in vivo nucleic acid delivery has been discovered (US 8,450,298; Love et al. 2010, PNAS 107, 1864-1869; WO2006/138380; Akinc et al. 2008, Nat Biotechnol 26, 561-569). Lipoids are obtained by reacting amine-containing compounds with aliphatic epoxides, acrylates, acrylamides, or aldehydes. The authors/inventors have provided synthetic procedures for obtaining lipidoid libraries and screening procedures for selecting useful compounds with utility in delivering nucleic acid to cells in vitro .

As is evident from the foregoing, much research and development has been done in the past on the provision of other nucleic acid molecules such as plasmid DNA, oligonucleotides, siRNAs or nucleic acid analogs. The supply of mRNA has not been investigated in great depth. Some authors have hypothesized that compounds and formulations that work well for DNA or siRNA delivery would work similarly for mRNA delivery. However, in contrast to plasmid DNA or siRNA, mRNA is a single-stranded molecule. Therefore, based on structural considerations alone, different requirements for compounds and formulations would be expected for delivery of mRNA versus delivery of aDn or siRNA.

The prior literature cited above describes the delivery of double-stranded nucleic acids such as plasmid DNA or siRNA to cells, but it is not known whether the methods and compounds described are capable of delivering single-stranded nucleic acids such as mRNA to cells. Notably, it has been previously observed that transfection of mRNA differs substantially from transfection of plasmid DNA into cells (Bettinger et al. 2001, Nucleic Acids Res, 29, 3882-91, Uzgün et al, 2011, Pharm Res, 28, 2223 -32).

In line with this, the present inventors found that when more than 100 members of a family of polymers disclosed in WO 2011/154331 are selected for their suitability in RNA delivery, preferably single-stranded RNA delivery such as mRNA , to cells, neither compound was useful for transfecting mRNA in a manner that results in expression of a gene encoded by the mRNA. On the contrary, all of these compounds are efficient in the delivery of plasmid DNA and/or siRNA. Therefore, the rules established for the delivery of double-stranded nucleic acids to cells do not apply a priori for single-stranded mRNA. The disclosure of WO 2011/154331 comprises chemically defined oligomers that are 2-60 units of oligo(alkyleneamino) acid units corresponding to the general formula 1HOOC-ZR-NH-[(CH ² ) ^b -NH] ^a -H , where Z is a methylene series or a variety of other groupings, R is a methylene or carboxyl residue, and a and b are independently integers of 1-7 or 2-7, respectively. Oligomers of this family comprise protonatable amino groups capable of exerting a so-called proton sponge effect and have been shown to be highly active in transfecting plasmid DNA and siRNA in vitro and in vivo. Importantly, WO 2011/154331 and associated scientific publications teach in great detail how sequence-defined oligomer/polymer libraries can be established from structural elements corresponding to the general formula HOOC-ZR-NH-[(CH ² ) ^b- NH] ^to -H.

The technical task underlying the present invention was therefore to provide a composition that is suitable for delivery of nucleic acids, and in particular RNA, preferably single-stranded RNA such as mRNA, with high efficiency to a cell or tissue. .

This task has been achieved by providing the embodiments as characterized in the claims and illustrated in more detail in the following general description and examples.

Surprisingly, it was found that copolymers containing a random/statistical arrangement of alternating length alkyleneamine repeat units in compositions for transfecting a cell with a nucleic acid, in particular RNA, preferably a single-stranded RNA such as mRNA, were consistently more effective than copolymers. polymers containing analogous arrangements of non-alternating length alkyleneamine repeating units.

Therefore, the invention provides, in a first aspect, a statistical copolymer comprising a plurality of repeating units (a) independently selected from repeating units of the following formulas (a1) and (a2):

a plurality of repeating units (b) independently selected from repeating units of the following formulas (b1) to (b4):

wherein the molar ratio of the sum of repeat units (a) to the sum of repeat units (b) is within the range of 0.7/1.0 to 1.0/0, 7, and

wherein one or more of the nitrogen atoms of the repeating units (a) and/or (b) contained in the copolymer may be protonated to provide a cationic copolymer.

In a further aspect, the invention provides a composition comprising a nucleic acid, in particular an RNA, preferably a single-stranded RNA such as mRNA, and the above copolymer.

In further aspects, the invention relates to pharmaceutical compositions comprising the compositions according to the invention. The invention also encompasses methods for the preparation of the copolymers according to the invention, as well as the compositions and pharmaceutical compositions according to the invention.

Still further aspects relate to the use of a composition according to the invention or a copolymer according to the invention to deliver a nucleic acid, in particular RNA, preferably a single-stranded RNA such as mRNA, to a target cell or tissue, and to a method for delivering a nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, to a cell comprising the step of contacting a composition with the cell according to the invention.

The copolymers according to the present invention combine shorter repeating units (a) with longer repeating units (b) in the form of a random copolymer, in particular a random copolymer, and in defined ratios. This arrangement of repeating units (a) and (b) has been found to provide unexpected advantages in terms of the suitability of the resulting copolymer as a vehicle for delivering a nucleic acid, in particular an RNA, preferably a single-stranded RNA such as mRNA, to a cell.

As indicated above, the copolymer according to the invention is a random copolymer comprising a plurality of repeating units (a) independently selected from repeating units of the following formulas (a1) and (a2):

— CH2—av - ^nh — (al)

(a2 ), _y

(a2 ), _and

a plurality of repeating units (b) independently selected from repeating units of the following formulas (b1) to (b4):

— CH2— CH2— CH2— NH—

(b1)

wherein the molar ratio of the sum of repeat units (a) to the sum of repeat units (b) is within the range of 0.7/1.0 to 1.0/0, 7, and

wherein one or more of the nitrogen atoms of the repeating units (a) and/or (b) contained in the copolymer may be protonated to provide a cationic copolymer.

The copolymer is a random copolymer, in which any repeating unit (a) and any repeating unit (b) are randomly distributed in the copolymer macromolecule. It is normally obtained from the copolymerization of a mixture of monomers that produce, during the polymerization reaction, the repeating units (a) with monomers that produce, during the polymerization reaction, the repeating units (b). Preferably, the copolymer is a random copolymer in which any repeating unit (a) and any repeating unit (b) are randomly distributed in the polymer macromolecule.

The copolymer according to the invention can be a linear, branched or dendritic copolymer. As the skilled reader will understand, a repeating unit of formula (a1), (b1) or (b3) with two valences (ie open bonds to neighboring units) leads to a linear propagation of the copolymer structure. Therefore, a linear copolymer of the invention comprises repeating units of formula (a1) and one or more types of repeating units of formulas (b1) and (b3), but without repeating units of formula (a2), (b2) or (b4). As will be further understood, the presence of a repeating unit of formula (a2), (b2) or (b4) with three valences provides a branch point in the copolymer structure. Therefore, a branched copolymer comprises one or more types of the repeating units of the formulas (a2), (b2) and (b4), and may further comprise one or more types of the repeating units of the formulas (a1), ( b1) and (b3).

The copolymer according to the invention comprises a plurality of repeating units (a) independently selected from repeating units of formulas (a1) and (a2) defined above, and a plurality of repeating units (b) independently selected from repeating units of formulas (b1) to (b4) defined above. Copolymers comprising a plurality of repeating units (a) independently selected from repeating units of formulas (a1) and (a2) defined above, and a plurality of repeating units (b) independently selected from repeating units of formulas (b1) and (b2) defined above.

It is also preferred that the copolymer according to the invention is a branched copolymer comprising one or more types of repeating units selected from repeating units (a2), (b2) and (b4), and optionally further comprising one or more types of the repeating units of formulas (a1), (b1) and (b3), and in particular a copolymer comprising repeating units of formula (a2) and one or more types of repeating units of formulas (b2) and ( b4), and optionally further comprising one or more kinds of the repeating units of formulas (a1), (b1) and (b3). In line with the above, a more preferred copolymer is therefore a branched copolymer comprising repeating units of formula (a2) and repeating units of formula (b2), and optionally further comprising one or more types of the repeating units of formulas (a1) and (b1).

In the copolymers according to the invention, the total number of repeating units (a) and repeating units (b) is usually 20 or more, preferably 50 or more, and more preferably 100 or more. Usually, the total number of the repeating units (a) and the repeating units (b) is 5,000 or less, preferably 2,500 or less, more preferably 1,000 or less, and particularly 500 or less.

Furthermore, it is preferred for the copolymers according to the invention that the repeating units (a) and (b) represent 80 mol% or more, more preferably 90 mol% or more of all the repeating units in the copolymer. Also preferred are copolymers in which the repeating units (a) selected from (a1) and (a2) and the repeating units (b) selected from (b1) and (b2) represent 80 mol% or more, more preferably 90 mol% or more of all repeating units in the copolymer. It is most preferred that all repeating units in the copolymer are repeating units (a) or (b), in particular that all repeating units in the copolymer are repeating units (a) selected from (a1) and ( a2) or repeating units (b) selected from (b1) and (b2).

The weight average molecular weight of the copolymer according to the present invention, as measured, for example, by gel permeation chromatography relative to linear standards of polyethylene oxide, generally ranges between 1,000 and 500,000 Da, preferably between 2,000 and 250,000 Da and more preferably 5,000 50,000 Da.

The terminal groups of the copolymer according to the invention normally comprise one or more types of groups (c) independently selected from the groups of formulas (c1) to (c3) below, preferably from the groups of formulas (c1) and (c2 ) next:

— CH2— CH2---CH2--- CH2--- NH2 (Cvi).

Preferably, the terminal groups in the copolymer consist of one or more types of groups (c) independently selected from groups of formulas (c1) to (c3) below, preferably from groups of formulas (c1) and (c2). As the person skilled in the art will understand, the number of terminal groups depends on the structure of the copolymer according to the invention. While a linear copolymer has only two terminal ends, larger numbers of terminal groups are contained in a branched group, particularly in a dendritic copolymer. As will be further understood, also one or more of the nitrogen atoms of the terminal groups (c) contained in the copolymer may be protonated to provide a cationic copolymer.

In the copolymer according to the invention, the molar ratio of the sum of the repeating units (a) to the sum of the repeating units (b) is within the range of 0.7/1.0 to 1, 0/0.7, and preferably within the range of 0.8/1.0 to 1.0/0.8. This molar ratio can be determined, for example, by NMR. Therefore, it will be understood that the ratio is usually determined for a plurality of macromolecules of the copolymer according to the invention, and normally indicates the global ratio of the sum of repeating units (a) with respect to the sum of repeating units (b). ) in the plurality of macromolecules.

As indicated above, one or more of the nitrogen atoms of the copolymer according to the invention may be protonated to result in a copolymer in cationic form, usually an oligocationic or polycationic form. It will be understood that primary, secondary or tertiary amino groups in repeating units (a) or (b) or in terminal groups (c) may act as proton acceptors, especially in water and aqueous solutions, including physiological fluids. Therefore, the copolymers of the present invention typically have an overall positive charge in aqueous solution at a pH of less than 7.5. An aqueous solution, as referred to herein, is a solution in which the solvent comprises 50% (vol./vol.) or more, preferably 80 or 90% or more, and most preferably 100% water. Furthermore, if the compositions according to the invention are in contact with a physiological fluid having a pH of less than 7.5, including, for example, blood and lung fluid, they normally contain repeating units (a) and (b) in which nitrogen atoms are protonated. The pKa values of the copolymers used in the compositions according to the invention can be determined by acid-base titration using an automated pKa titrator. The net charge at a given pH value can then be calculated, for example, from the Henderson-Hasselbach equation. Any load can be shared between several of the basic centers and cannot necessarily be attributed to a single point. Normally, in solutions at physiological pH, the copolymers used in the compositions according to the invention comprise repeating units with amino groups in the protonated state and repeating units with amino groups in the unprotonated state.

However, as the skilled reader will understand, the copolymers according to the invention, as well as the compositions according to the invention, can also be provided as a dry salt form containing the copolymer in cationic form.

As will be further understood, counterions (anions) to the positive charges of protonated amino groups in compositions according to the invention comprising the copolymer and nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, are normally provided by anionic moieties contained in the nucleic acid. If positively charged groups are present in excess compared to anionic moieties in the nucleic acid, the positive charges may be balanced by other anions, particularly those normally found in physiological fluids, such as Cl- or HCO3-.

In line with the above, a preferred copolymer according to the invention is a random copolymer, in which

80 mol% or more of all repeating units, more preferably all repeating units, are made up of

a plurality of repeating units (a) independently selected from repeating units of the following formulas (a1) and (a2):

a plurality of repeating units (b) independently selected from repeating units of the following formulas (b1) and (b2):

wherein the molar ratio of the sum of repeat units (a) to the sum of repeat units (b) is within the range of 0.7/1.0 to 1.0/0, 7, more preferably within the range of 0.8/1.0 to 1.0/0.8;

in which the end groups of the copolymer are formed by

groups (c) independently selected from groups of formulas (c1) and (c2):

----- CH2— CH2— NHs

(C1)

—CHj—CH2—CH2— ^nh 2 (c2);

Y

wherein one or more of the nitrogen atoms of the repeating units (a) and/or (b) and/or of the end groups (c) contained in the copolymer may be protonated to provide a cationic copolymer. It is further preferred that the copolymer is a branched copolymer, comprising units (a2) and (b2), optionally together with units (a1) and/or (b1).

The copolymers according to the invention can conveniently be prepared by processes analogous to those known for the preparation of polyalkylenimines, such as branched or linear polyethyleneimine (PEI). It will be understood that the monomers used for the production of the copolymers will have to be adjusted accordingly. In the context of the present invention, it has been found that the monomers can conveniently be reacted quantitatively, so that the ratio of units (a) and (b) in the copolymer can be adjusted by adjusting the ratio of monomers accordingly in the monomer mixture subjected to polymerization.

While polyethyleneimine, also called polyaziridine, can be prepared, for example, by ring-opening polymerization of aziridine, the copolymers according to the invention can be prepared by ring-opening polymerization of a monomer mixture comprising or consisting of aziridine, azetidine and, where applicable, pyrrolidine, or, in preferred embodiments, of aziridine and azetidine. It will be understood that the expression "where applicable" refers to the presence or absence of repeating units (b3) and (b4) or terminal groups (c3) that would be formed by the pyrrolidine. Ring-opening polymerization of unsubstituted cyclic amines usually leads to branched copolymers. Linear copolymers according to the invention may be prepared, for example, by polymerization of suitable N-substituted aziridines, N-substituted azetidines and N-substituted pyrrolidines, or N-substituted aziridines and N-substituted azetidines, which may be followed by, for example, a hydrolytic cleavage of N-substituents attached to the resulting polyalkyleneimine chain, eg, in analogy to the procedure published in Katrien F. Weyts, Eric J. Goethals, New synthesis of linear polyethyleneimine, Polymer Bulletin, January 1988, volume 19, issue 1, p. 13-19.

For the preparation of a dendrimer (or dendritic copolymer), synthesis strategies can be applied analogously to those known for the production of polyethyleneimine or polypropyleneamine dendrimers. Polypropylenimine dendrimers can be synthesized from acrylonitrile building blocks using a repetitive sequence of a Michael addition to a primary amine, followed by heterogeneously catalyzed hydrogenation (Newkome and Shreiner Poly(amidoamine), polypropylenimine, and related dendrimers and dendrons possessing different 1^-2 branching motifs: An overview of the divergent procedures. Polymer 49 (2008) 1-173; De Brabander-Van Den Berg et al. Large-scale production of polypropylenimine dendrimers, Macromolecular Symposia (1994) 77 (1) 51 -62). Polyethyleneimine dendrimers can be produced using a repetitive sequence of a Michael addition of a vinyl bromide building block to a primary amine followed by a conversion of alkyl bromide to amine using a Gabriel amine synthesis method (Yemul & Imae, Synthesis and characterization of poly(ethyleneimine) dendrimers, Colloid Polym Sci (2008) 286:747-752). Therefore, the person skilled in the art will be able to produce not only dendrimers with strictly alternating layers of, for example, propylenimines and ethyleneimines. Similarly, generations of layered dendrimers can be generated comprising or consisting of random compositions of repeating units of formula (a2), (b2) and (b4) and preferably repeating units (a2) and (b2).

The ring-opening polymerization of aziridine and azetidine, or of aziridine, azetidine and pyrrolidine, can be carried out in solution, for example in water. The total monomer concentration is not particularly limited, typical concentrations range from 10% w/w to 80% w/w, preferably 30% w/w and 60% w/w. Polymerization is normally initiated by protons, so it is preferred to add a Br0nsted acid, in particular a mineral acid such as sulfuric acid, to the reaction system. In general, small amounts of acid, such as 0.001 to 0.01 equivalents, based on total monomer concentration, are sufficient. The reaction proceeds at convenient rates, for example in the temperature range 50 to 150°C, in particular 90 to 140°C. In these ranges, higher molecular weight copolymers are usually at higher temperatures, and lower molecular weight copolymers at lower temperatures.

Nucleic acid

As indicated above, a central aspect of the invention is a composition comprising a nucleic acid, in particular an RNA, preferably a single-stranded RNA such as mRNA, and the above copolymer according to the invention.

The term "nucleic acid" encompasses all forms of naturally occurring types of nucleic acids, as well as chemically and/or enzymatically synthesized nucleic acids, and also encompasses nucleic acid analogs and nucleic acid derivatives such as, for example, locked nucleic acids (LNA), peptide nucleic acids (PNA), oligonucleoside thiophosphates and phosphotriesters, morpholino oligonucleotides, cationic oligonucleotides (US6017700 A, WO/2007/069092), substituted ribooligonucleotides or phosphorothioates. Furthermore, the term "nucleic acid" also refers to any molecule that comprises nucleotides or nucleotide analogs. There are no limitations with respect to the sequence or size of a nucleic acid comprised in the composition of the present invention. Nucleic acid is predominantly defined by the biological effect to be achieved on the biological target to which the composition of the present invention is delivered. For example, in the case of a nucleic acid or gene therapy application, the nucleic acid or nucleic acid sequence may be defined by the gene or gene fragment to be expressed or by the intended replacement or repair of a defective gene or any gene target sequence or by the target sequence of a gene to be inhibited, inactivated or down-regulated.

Preferably, the term "nucleic acid" refers to oligonucleotides or polynucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). As far as RNA is concerned, in principle, any type of RNA can be used in the context of the present invention. In a preferred embodiment, the RNA is a single-stranded RNA. The term "single-stranded RNA" means a single consecutive strand of ribonucleotides in contrast to RNA molecules in which two or more separate strands form a double-stranded molecule due to hybridization of the separate strands. The term "single-stranded RNA" does not exclude that the single-stranded molecule itself forms double-stranded structures such as loops, secondary or tertiary structures.

The term "RNA" covers RNA that encodes an amino acid sequence, as well as RNA that does not encode an amino acid sequence. It has been suggested that more than 80% of the genome contains functional DNA elements that do not code for proteins. These non-coding sequences include regulatory DNA elements (binding sites for transcription factors, regulators, and co-regulators) and sequences that encode transcripts that are never translated into proteins. These transcripts, which are encoded by the genome and are transcribed into RNA but not translated into protein, are called non-coding RNAs (ncRNAs). Thus, in one embodiment, the RNA is non-coding RNA. Preferably, the antisense RNA is a single-stranded molecule. Studies show that ncRNAs are critical players in gene regulation, maintenance of genomic integrity, cell differentiation and development, and are misregulated in various human diseases. There are different types of ncRNA: short (20-50 nt), medium (50-200 nt), and long (>200 nt) ncRNAs. Short ncRNA includes microRNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), and transcriptional initiator RNAs (tiRNAs). Examples of medium ncRNAs are small nuclear RNAs (snRNAs), small nucleolar RNAs (snRNAs), transfer RNAs (tRNAs), transcription start site-associated RNAs (saRNAs), promoter-associated small RNAs (PASRs), and transcribed downstream of the promoter (PROMPT). Long noncoding RNAs (nclRNAs) include long intergenic noncoding RNA (lincRNA), antisense nclRNA, intronic ncl, ultraconserved transcribed RNAs (T-UCR), and others (Bhan A, Mandal SS, ChemMedChem. Mar 26, 2014. doi : 10.1002/cmdc.201300534). Of the non-coding RNAs mentioned above, only siRNA is double-stranded. Therefore, since in a preferred embodiment the antisense RNA is single-stranded, it is preferred that the antisense RNA is not siRNA. In another embodiment, the RNA is a coding RNA, ie, an RNA that encodes an amino acid sequence. Such RNA molecules are also called mRNA (messenger RNA) and are single-stranded RNA molecules. Nucleic acids may be prepared by chemical and enzymatic synthesis methodology known to one of skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof. The oligonucleotides or polynucleotides may optionally comprise non-natural nucleotides and may be single-stranded or double-stranded or triple-stranded. "Nucleic acid" also refers to sense and antisense oligo- or polynucleotides, ie, a nucleotide sequence that is complementary to a specific nucleotide sequence in a DNA and/or RNA.

Preferably, the term "nucleic acid" in the context of the present invention refers to RNA, more preferably to single-stranded RNA, in particular to mRNA, and most preferably to modified mRNA.

Messenger RNAs (mRNA) are copolymers that are formed from nucleoside phosphate structural elements mainly with adenosine, cytidine, uridine and guanosine as nucleosides, which as intermediate carriers carry the genetic information of DNA in the cell nucleus to the cytoplasm, where it is translates into proteins. Therefore, they are suitable as alternatives for gene expression.

In the context of the present invention, mRNA is to be understood to mean any polyribonucleotide molecule which, if it enters the cell, is suitable for the expression of a protein or fragment thereof or can be translated into a protein or fragment thereof. The term "protein" herein encompasses any type of amino acid sequence, ie, chains of two or more amino acids that are each linked through peptide bonds, and also includes peptides and fusion proteins.

mRNA contains a ribonucleotide sequence that encodes a protein or protein fragment whose function in or near the cell is necessary or beneficial, for example, a protein whose missing or defective form is a trigger for disease or condition, the provision of which can moderate or prevent a disease or condition, or a protein that can promote a process that is beneficial to the body, in or near a cell. The mRNA may contain the sequence for the entire protein or a functional variant thereof. Furthermore, the ribonucleotide sequence may encode a protein that acts as a factor, inducer, regulator, stimulator or enzyme, or a functional fragment thereof, where this protein is one whose function is necessary to remedy a disorder, in particular a metabolic disorder. or to initiate processes in vivo such as the formation of new blood vessels or tissues. Herein, functional variant is understood to mean a fragment which, in the cell, can carry out the function of the protein whose function in the cell is required or the lack or defective form thereof is pathogenic. In addition, the mRNA may also have additional functional 3' or 5' non-coding regions and/or regions. The 3' and/or 5' non-coding regions may be the regions that naturally flank the protein coding sequence or artificial sequences that contribute to RNA stabilization. Suitable sequences for this in each case can be determined by those skilled in the art by routine experiments.

In a preferred embodiment, the mRNA contains an m7GpppG cap, an internal ribosome entry site (IRES) and/or a polyA tail at the 3' end in particular to enhance translation. The mRNA may have additional regions that promote translation.

In a preferred embodiment, the mRNA is an mRNA that contains a combination of modified and unmodified nucleotides. Preferably, it is an mRNA containing a combination of modified and unmodified nucleotides as described in WO2011/012316. The mRNA described therein is reported to show increased stability and decreased immunogenicity. In a preferred embodiment, in an mRNA modified in this type, 5 to 50 % of the cytidine nucleotides and 5 to 50 % of the uridine nucleotides are modified. Nucleotides containing adenosine and guanosine may be unmodified. Adenosine and guanosine nucleotides may be unmodified or partially modified, and are preferably present in unmodified form. Preferably 10 to 35% of the cytidine and uridine nucleotides are modified, and particularly preferably the content of the modified cytidine nucleotides is in a range of 7.5 to 25% and the content of the modified uridine nucleotides in a range of 7.5 to 25%. It has been found that, in fact, a relatively low content, eg only 10% each, of modified cytidine and uridine nucleotides can achieve the desired properties. It is particularly preferred that the modified cytidine nucleotides are 5-methylcytidine residues and the modified uridine nucleotides are 2-thiouridine residues. Most preferably, the content of the modified cytidine nucleotides and the content of the modified uridine nucleotides is 25%, respectively.

In another preferred embodiment, the mRNA may be combined with target binding sites, targeting sequences and/or with micro-RNA binding sites, to allow activity of the desired mRNA only in relevant cells. In a further preferred embodiment, the RNA may be combined with micro-RNAs or shRNAs downstream of the 3' polyA tail.

Furthermore, the term "nucleic acid(s)" may refer to DNA or RNA or hybrids thereof or any modifications thereof that are known in the art (see, for example, US 8278036 , WO 2013/052523, WO 2011/012316, US 5525711, US 4711955, US 5792608 or EP 302175, (Lorenz et al.

2004, Bioorg Med Chem Lett, 14, 4975-4977; Soutschek et al. 2004, Nature, 432, 173-178) for examples of modifications). Such nucleic acid molecule(s) are single-stranded or double-stranded, linear or circular, natural or synthetic, and without any size limitation. For example, the nucleic acid molecule(s) may be genomic DNA, cDNA, mRNA, antisense RNA, ribozyme or small interfering RNAs (siRNAs), microRNAs, antagomirs, or short hairpin RNAs (shRNAs), tRNA or long double-stranded RNA or a DNA construct encoding such RNA or chimeraplasts (Colestrauss et al. 1996, Science, 273, 1386-1389), or aptamers, clustered regularly interspaced short palindromic repeats ("CRISPR" for cleavage of RNA-guided site-specific DNA) (Cong et al. 2013, Science, 339, 819-823), or RNA and DNA. Said nucleic acid molecule(s) may be in the form of plasmids, cosmids, artificial chromosomes, viral DNA or RNA, bacteriophage DNA, single-stranded or double-stranded coding and non-coding RNA (mRNA) and oligonucleotide(s). ), including any of the prior art modifications to the sugar backbone and/or bases as described above and 3' or 5' modifications. In a particularly preferred embodiment, the nucleic acid is RNA, more preferably mRNA or siRNA.

The nucleic acid(s) may contain a nucleotide sequence that encodes a polypeptide to be expressed in a target cell. Methods well known to those of skill in the art can be used to construct recombinant nucleic acid molecules; see, for example, the techniques described in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (2001) NY and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1989 ).

In a preferred embodiment, said nucleic acid is a therapeutically or pharmaceutically active nucleic acid including all types and modifications of nucleic acid listed above and those known to a person skilled in the art that can have a therapeutic or preventive effect. In general, therapeutic or preventive effects can be achieved by the interaction of the nucleic acid with cellular molecules and organelles. Such interaction alone can activate, for example, the innate immune system, as is the case with certain CpG oligonucleotides and sequences designed to specifically interact with toll-like and other extra- or intracellular receptors. Furthermore, the uptake or introduction of nucleic acids into cells may be aimed at leading to the expression of nucleotide sequences such as genes comprised in the nucleic acid, it may be aimed at down-regulation, silencing or inactivation of gene expression. endogenous as a consequence of the intracellular presence of an introduced exogenous nucleic acid, or may be intended for modification of endogenous nucleic acid sequences such as repair, excision, insertion or exchange of selected bases or entire stretches of endogenous nucleic acid sequences, or it may be intended to interfere with virtually any cellular process as a consequence of the intracellular presence and interaction of an introduced exogenous nucleic acid. The overexpression of introduced exogenous nucleic acids may be intended to compensate or complement endogenous gene expression, in particular in cases where an endogenous gene is defective or silent, leading to an absent, insufficient or defective or dysfunctional product of gene expression, as is the case with many metabolic and hereditary diseases such as cystic fibrosis, hemophilia or muscular dystrophy, to name a few. Overexpression of introduced exogenous nucleic acids may also be intended for the expression product to interact or interfere with any endogenous cellular processes such as regulation of gene expression, signal transduction and other cellular processes. Overexpression of introduced exogenous nucleic acids may also be intended to give rise to an immune response in the context of the organism in which a transfected or transduced cell resides or is caused to reside. Examples are the genetic modification of antigen presenting cells such as dendritic cells to present an antigen for vaccination purposes. Other examples are the overexpression of cytokines in tumors to elicit a tumor-specific immune response. In addition, the overexpression of introduced exogenous nucleic acids may also be aimed at generating genetically modified cells. transiently in vivo or ex vivo for cell therapies such as modified T cells or precursor or stem cells or others for regenerative medicine.

Downregulation, silencing, or inactivation of endogenous gene expression for therapeutic purposes can be achieved, for example, by RNA interference (iaRn), with ribozymes, antisense oligonucleotides, tRNA, long double-stranded RNA where such downregulation can be sequence-specific or non-specific and can also lead to cell death as is the case when long double-stranded RNAs are introduced into cells. Down-regulation, silencing, or inactivation of pre-existing or endogenous gene expression may be useful in the treatment of acquired, hereditary, or spontaneous diseases, including viral infections and cancer. It can also be envisaged that the introduction of nucleic acids into cells can be practiced as a preventative measure to prevent, for example, viral infection or neoplasms. Down-regulation, silencing, or inactivation of endogenous gene expression can be exerted at the transcriptional level and at the translational level. Multiple mechanisms are known to those skilled in the art and include, for example, epigenetic modifications, changes in chromatin structure, selective binding of transcription factors by the introduced nucleic acid, hybridization of the introduced nucleic acid with complementary sequences in genomic DNA, mRNA or other RNA species by base pairing, including unconventional base pairing mechanisms such as triple helix formation. Similarly, gene repair, base or sequence changes can be achieved at the genomic level and at the mRNA level, including exon skipping. Base or sequence changes can be achieved, for example, by RNA-guided site-specific DNA cleavage, by splicing mechanisms that take advantage of trans-splicing, trans-splicing ribozymes, chimeraplasts, splicing and spliceosome-mediated trans e-splicing of RNA either by exploiting redirected or group II introns, or by exploiting virus-mediated insertional mutagenesis or by exploiting targeted genomic insertion using prokaryotic, eukaryotic, or viral integrase systems. Since nucleic acids are the carriers of the building plans of living systems and since they participate in many cellular processes directly and indirectly, theoretically, any cellular process can be influenced by the introduction of nucleic acids into cells from the outside. Notably, this introduction can be carried out directly in vivo and ex vivo in cell or organ culture followed by transplantation of the organs or cells so modified into a recipient. The complexes of the present invention with nucleic acids as active agents may be useful for all the purposes described above.

Composition

As disclosed above, the composition according to the invention comprises a nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, and the copolymer according to the invention.

The invention also encompasses a composition consisting of the nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, and the copolymer according to the invention. However, the composition may also comprise additional components, for example, components for lipid formulation and/or components that exert an effector function during delivery of the nucleic acid to and within a cell. Typically, the combined weights of the nucleic acid and the copolymer according to the invention represent 50% by weight or more, preferably 70% by weight or more, of the total weight of the composition.

It will be understood that the compositions according to the invention generally provide an association of the nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, with the copolymer and optional additional components, which are associated into a finite entity, stable enough to maintain the association of a significant proportion of said components to reach a biological target or the vicinity of a biological target during an application, eg, during a desired route of delivery of nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA.

Due to the presence of the protonatable amino groups in the copolymer according to the invention, this copolymer may comprise cationic charges in the repeating units of formula (a) and/or (b), so that the copolymer forms a cation, normally a oligo- or polycation containing a plurality of cationic moieties, in the presence of protons, for example, in water or aqueous solutions, or in the presence of a proton-donating acid. Therefore, preferably, the composition according to the invention contains or consists of a complex of nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, and a copolymer according to the invention which is a cationic copolymer. It will be understood that a cationic copolymer and an anionic nucleic acid are generally associated by electrostatic interaction in such a complex. However, other attractive interactions may also participate in the stabilization of the complex, including hydrogen bonds and covalent bonds.

In the compositions of the present invention, the copolymer and nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, are normally contained, for example, in a weight ratio of copolymer/weight of nucleic acid (w/w) of 0.25/1 - 50/1, preferably 0.5/1 - 30/1, more preferably 1/1 - 20/1.

More preferably, in cases where the composition contains a complex of the nucleic acid, particularly RNA, preferably single-stranded RNA such as mRNA, and a cationic copolymer according to the invention, they may Relative ratios of the copolymer and nucleic acid, in the compositions of the invention, may be selected in consideration of the degree of mutual charge neutralization. In nucleic acid, particularly RNA, preferably single-stranded RNA such as mRNA, the supply with complexes of the nucleic acid, with a cationic copolymer, in general, quantities of the cationic copolymer are mixed with a given quantity of the nucleic acid, leading to al minus a charge neutralization of the negative charges of the nucleic acid, preferably to an overcompensation of the negative charges of the nucleic acid.

Suitable ratios between cationic copolymer and nucleic acid, particularly RNA, preferably single-stranded RNA such as mRNA, can be determined by gel retardation assays, fluorescence quenching methods such as the ethidium bromide quenching/shift assay, by measurements the size and zeta potential of the particles. Useful ratios between the copolymer and the nucleic acid, particularly RNA, preferably single-stranded RNA such as mRNA, are generally characterized by at least partial, preferably complete retardation of the nucleic acid comprised in the complex with the cationic copolymer when subjected to electrophoresis in an agarose gel, by high degree fluorescence quenching of dyes such as ethidium bromide, RiboGreen or YOYO when intercalated into nucleic acids or by the formation of (nano)particles by mixing the copolymer and nucleic acid.

For compositions containing the chemically well-defined cationic copolymers of the present invention, the calculated N/P ratio is a suitable factor for choosing and defining the relative ratios of the copolymer and the nucleic acid, particularly RNA, preferably single-stranded RNA such as mRNA. The N/P ratio designates the molar ratio of the protonatable nitrogen atoms in the repeating units (a) and (b) of the copolymer of the present invention to the phosphate groups of the nucleic acid in the composition of the present invention. The N/P ratio is an established parameter for the characterization of such nucleic acid complexes with cationic carriers. The skilled reader will understand that nitrogen atoms in amino groups linking repeating units (a) and (b) to neighboring repeating units and terminal amino groups are considered to be protonatable nitrogen atoms. On the other hand, nitrogen atoms in amide bonds (which are generally not present in the copolymers of the invention), would not count as protonatable nitrogen atoms. For compositions of the present invention containing a cationic copolymer of the invention and nucleic acid, particularly RNA, preferably single-stranded RNA such as mRNA, the N/P ratio can be conveniently calculated, for example, according to the formula

where wp is the weight of the copolymer in grams, n is the number of protonatable amino groups per repeating unit, Mwp is the average molecular weight of the repeating units (a) and (b) contained in the copolymer, wna is the weight of nucleic acid in grams and Mbase is the average molecular weight of a nucleotide in the nucleic acid, eg 346 g/mol in the case of a Rn .

Specifically for the copolymers of the present invention, n is 1. Mwp can be calculated based on the ratio of polymerized repeating units, eg, determined by NMR, and their respective molecular weights. For example, in a branched copolymer containing polymerized units (a2) and (b2) with molecular weights of 42 g/mol (a2) and 56 g/mol (b2), respectively, in a molar ratio of (a)/( b) from 0.9/1.0, Mwp is calculated as

0.9X42+ -1.0X56

In binary complexes according to the invention consisting of the copolymer according to the invention and nucleic acid, particularly RNA, preferably single-stranded RNA such as mRNA, for the delivery of nucleic acid according to the invention, relative amounts of the copolymer to acid should preferably be used. nucleic acids that provide an N/P ratio that results in a positive zeta potential of the final binary composition. In the context of the present invention, for binary compositions of the present invention, N/P ratios of 1 to 100 are preferred, N/P ratios of 3 to 60 are more preferred, and N/P ratios of 4 to 60 are most preferred. 44.

The composition of the invention optionally comprises other components, in particular components that can exert an effector function during the delivery of nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, to and within a cell. Such components may be, but are not limited to, polyanions, lipids, polycations other than the copolymers of the present invention including cationic peptides, protective oligomers or polymers, poloxamers (also known as pluronics), poloxamines, targeting ligands, endosomolytic agents, signal and cell-penetrating peptides, magnetic and non-magnetic nanoparticles, RNase inhibitors, fluorescent dyes, radioisotopes or contrast agents for medical imaging. The term "effector function" encompasses any function that supports the achievement of an effect. biological target of a nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, of the composition to or in a biological target or in the vicinity of a biological target. For example, nucleic acid delivery compositions have been formulated to comprise antisense nucleic acids or non-nucleic acid polyanions as fillers (Kichler et al., 2009, 2005, J Gene Med, 7, 1459-1467). Such fillers are suitable for reducing the dose of a nucleic acid having an intended biological effect while maintaining the extent or degree of that effect obtained at a higher dose of nucleic acid in the absence of such filler. Non-nucleic acid polyanions have also been used to obtain prolonged in vivo gene expression at reduced toxicity (Uchida et al. 2011, J. Control Release, 155, 296-302). The compositions of the present invention may also comprise cationic, anionic or neutral lipids as is the case in lipopolyplexes (Li and Huang in "Nonviral Vectors for Gene Therapy", Academic Press 1999, chapter 13, 295-303). In addition, the compositions of the present invention may comprise oligo or polycations other than the copolymers of the present invention. Such additional oligo- or polycations may be useful in achieving a desired degree of compaction of a nucleic acid or, in the case of polycationic peptides, may have a nuclear localization signal function as previously described (Ritter et al. 2003, J Mol Med, 81, 708-717). Protective polymers such as polyethylene glycol (PEG) may also be comprised in the compositions of the present invention and are often used to stabilize polyplexes and lipoplexes against aggregation and/or unwanted interactions in a biological environment (opsonization), e.g. example, interactions with serum components, blood cells or extracellular matrix. Protection may also be suitable to reduce the toxicity of compositions comprising nucleic acids (Finsinger et al. 2000, Gene Ther, 7, 1183-1192). Protective polymers such as PEG can be directly covalently coupled to the copolymers of the present invention. The coupling can be achieved, for example, to terminal groups (c), or to an amino group of the repeating units of formulas (a1), (b1) or (b3).

Polyvinyl derivatives such as PVP and poloxamers have been found to be useful in enhancing transfection following intramuscular injection (Mumper et al. 1996, Pharm Res, 13, 701-709, Lemieux et al. 2000, Gene Ther, 7, 986 991) and therefore may be usefully encompassed in the compositions of the present invention.

Targeting ligands including antibodies comprised in nucleic acid delivery compositions are useful for preferential and enhanced transfection of target cells (Philipp and Wagner in "Gene and Cell Therapy - Therapeutic Mechanisms and Strategy," 3rd edition, chapter 15. CRC Press, Taylor & Francis Group LLC, Boca Raton 2009). A targeting ligand can be any compound that confers target recognition and/or target binding function to the compositions of the present invention in a direct or indirect manner. In more general terms, a target is a distinct biological structure to which a targeting ligand can specifically bind through molecular interaction and where such binding will ultimately lead to the preferential accumulation of the nucleic acid comprised in the composition in a target tissue and /oao in a target cell. Similar to the pEg chains, targeting ligands can be coupled, for example, to terminal groups (c), or to an amino group of the repeating units of formulas (a1), (b1) or (b3).

In addition, endosomolytic agents such as endosomolytic peptides (Plank et al. 1998, Adv Drug Deliv Rev, 34, 21-35) or any other compound that is suitable for enhancing endosomal release of an endocytosed nucleic acid are useful components of compositions of the present invention. Similarly, cell-penetrating peptides (in another context also known as protein transduction domains) (Lindgren et al. 2000, Trends Pharmacol Sci, 21, 99-103) may be useful components of the composition of the present invention. to mediate the intracellular delivery of a nucleic acid. The so-called TAT peptide is within this class and also has nuclear localization function (Rudolph et al. 2003, J Biol Chem, 278, 11411-11418).

The magnetic nanoparticles that may be comprised in the compositions of the present invention are useful for physical targeting of delivery by magnetic force and for dramatically improving the efficiency of nucleic acid transfer, a mechanism also known as magnetofection (EP1297169; Plank et al., 2011, Adv Drug Deliv Rev, 63, 1300-1331). Similarly, a composition of the present invention may also be a non-magnetic or magnetic microbubble used for physical enhancement and targeting of nucleic acid delivery via ultrasound and optional application of a magnetic field (Holzbach et al. 2010 , J Cell Mol Med, 14, 587-599, Vlaskou et al., 2010, Adv Funct Mater, 20, 3881-3894). Quantum dots (Zintchenko et al. 2009, Mol Ther, 17, 1849-1856), radioactive tracers, and medical imaging contrast agents can be advantageously used to track the delivery of nucleic acids and to determine the biodistribution of compositions for the supply of nucleic acids. In summary, numerous effectors for nucleic acid delivery have been described and may be useful components in compositions comprising nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA and a copolymer according to the invention.

Those skilled in the art are well aware that a great degree of flexibility exists with respect to the amount of substance of each component comprised in the composition according to the present invention. For example, the so-called monomolecular binary polyplexes for plasmid DNA have been described, where the composition consists of nanoparticles formed after mixing the polycation and the plasmid DNA that comprise exactly a single molecule of plasmid DNA and as many polycation molecules as are required for the charge neutralization or charge overcompensation (positive versus negative) (DeRouchey et al. 2006, J Phys Chem B.

110(10):4548-54). For PEI-DNA, complexes at N/P ratios that are often used in transfections, were found by fluorescence correlation spectroscopy to contain on average 3.5 (+/-1) plasmid DNA molecules and 30 PEI molecules. , while approximately 86% of the PEI molecules used to prepare the complexes were in the free form (Clamme et al. 2003, Biophys J 84, 1960-1968). At the other extreme, aggregate complexes of PEI and plasmid DNA, putatively comprising a large number (tens to hundreds) of the component molecules, were found to have better transfection performance than small, discrete PEI-DNA nanoparticles (Ogris et al. al 1998, Gene Ther, 5, 1425-1433; Ogris et al 2001, Aa PS PharmSci, 3, E21). Therefore, the composition according to the present invention can be a particle, in particular a nanoparticle comprising a few nucleic acid molecules, in particular RNA, preferably single-stranded RNA such as mRNA, but can also be a macroscopic object such as a precipitate. or a dry powder comprising a large number of nucleic acid molecules, in particular RNA, preferably single-stranded RNA such as mRNA. In summary, the compositions of the present invention are characterized by the input rates of their components prior to self-assembly. Typical input w/w ratios of the individual components relative to the nucleic acid component, in particular RNA, preferably single-stranded RNA such as mRNA, are between 1 and 50. The N/P ratio is a suitable measure of the input ratio for binary compositions containing the copolymer of the invention and the nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA when the copolymer is chemically well defined. As indicated above, values for the N/P ratio of 1 to 100 are preferred, more preferred are N/P ratios of 3 to 60, and most preferred are N/P ratios of 4 to 44.

If the composition of the present invention comprises additional components, an assignment of an N/P ratio may be ambiguous. In this case, suitable input ratios are determined by experiments including, but not limited to, gel retardation assays, fluorescence quenching assays such as the ethidium bromide quenching/shift assay, by measurements of particle size particle size and zeta potential of the particles and by functional assays such as transfection assays as described herein. In ternary complexes comprising additional polyanion or protective polymers, the ratio of net charge (positive to negative) may be less than 1 and the zeta potential may be neutral or negative.

The composition of the invention can be produced as described below. Typically, nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA with a negative charge, and the copolymers of the present invention preferably in cationic form can self-assemble when contacted especially in a suitable solvent. After the self-assembly process, the composition of the present invention can be separated from any unincorporated components and in the same step the suspension medium can be replaced by centrifugation or by ultrafiltration or size exclusion chromatography or dialysis or any related method. The stoichiometry of the components of the composition of the present invention, purified or unpurified, can be determined by a variety of analytical methods including spectroscopic methods such as UV/VIS spectrometry or fluorescence correlation spectroscopy (DeRouchey et al. 2006, J Phys Chem B. 110(10):4548-54), by orthogonal fluorescence or radioisotope labeling of the individual components, by NMR and IR spectroscopy or chromatographic analysis and quantification after disassembly of the composition. Disassembly can be achieved, for example, by the addition of excess polyanion such as heparin as described herein or chondroitin sulfate or by the addition of sodium dodecyl sulfate.

The present invention also relates to a method for producing the composition of the invention. Copolymers according to the present invention can be produced and purified as described herein. The copolymers can be stored in aqueous solution or in dry form, such as a dry powder, in which case they can be redissolved in aqueous medium, preferably water, before producing the composition. The pH of the solution is adjusted to neutral or slightly acidic (up to pH 4.5) with an acid, preferably hydrochloric or citric acid, if required. In the case of RNA, preferably single-stranded RNA such as mRNA, the nucleic acid being comprised in the composition, it is preferred that the pH be adjusted to about 4.5 to 5.5, preferably about 4.9 to 5, 1, more preferably about 5.0. Nucleic acids are produced and purified according to the state of the art well known to those skilled in the art. The nucleic acid is provided as a solution in aqueous medium, preferably water. Optionally, either the copolymer or the RNA as nucleic acid or both are chemically linked with effector molecules such as targeting ligands, signal peptides, cell-penetrating peptides, endosomolytic substances or protective polymers. However, depending on the chemical nature of the effector molecules, they may not need to be chemically bonded, but may be incorporated into the composition of the present invention by self-assembly based on non-covalent bonding, i.e., electrostatic, hydrophobic, or bonding interaction. Van-der-Waals with any of the other components of the composition. For this purpose, it may be advantageous to adjust the ionic strength, the type of counterion, the pH or the organic solvent content of individual component solutions.

As an alternative to the mixing procedure described above, the nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, and the copolymer component can be mixed with an automated micromixing device as described, for example, by Hirota et al. (Hirota et al. 1999, Biotechniques, 27, 286-290) or Kasper et al. (Kasper et al. 2011, Eur J Pharm Biopharm, 77, 182-185) or by targeting microfluidics as reviewed by Xuan et al. (Xuan et al. 2010, Microfluidics and nanofluidics, 9, 1-16).

The composition of the present invention comprising nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, can then be prepared by self-assembly after mixing the component solutions. Self-assembly can be achieved by hand mixing using pipetting and vortexing/agitation or by using an automated micromixing device as described, for example, by Hirota et al. (Hirota et al. 1999, Biotechniques, 27, 286-290) or Kasper et al. (Kasper et al. 2011, Eur J Pharm Biopharm, 77, 182-185) or by microfluidic targeting as reviewed by Xuan et al. (Xuan et al. 2010, Microfluidics and nanofluidics, 9, 1-16). If the composition of the present invention comprises additional components in addition to nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, and the copolymer of the present invention, sequential mixing may be required. In this case, any additional components may be added after self-assembly of the copolymer and nucleic acid, or may be added to either prior to mixing. The most suitable sequence of mixing steps will depend on the chemical nature of the additional components. For example, if the additional component is negatively charged, it may be most suitable to add it to the nucleic acid component prior to mixing with the copolymer or to a preformed complex of the copolymer and nucleic acid, where the copolymer of the present invention is present in excess. in terms of the ratio of positive charges to the sum of the negative charges of the nucleic acid and the additional anionic component. Conversely, if the additional component is cationic, it may be most suitable to add it to the copolymer of the invention before mixing it with the nucleic acid. Or it can be used in a stoichiometry to partially neutralize the negative charges of the nucleic acid followed by mixing with the copolymer solution of the present invention. In the case of nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA comprising complexes for magnetofection, salt-induced colloidal aggregation has been shown to be a suitable means of preparing compositions comprising a nucleic acid, a polycation or a cationic lipid and magnetic particles (EP1297169). In the special case of nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, the component being a cationic oligonucleotide, a polyanion may be used to self-assemble the copolymer of the present invention with the nucleic acid. In this case, the copolymer of the present invention is mixed with the cationic oligonucleotide followed by mixing with the polyanion. It will be apparent to those skilled in the art that numerous formulation options are available to obtain the composition of the present invention. The concentrations of the individual components are chosen according to the intended use of the composition of the present invention. Relevant parameters are the final concentration of the nucleic acid, in particular RNA, preferably single-stranded RNA as mRNA component and the ratio of components as described above. For delivery of nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA in cell culture, final nucleic acid concentrations of between 1 and 100 µg/ml are generally preferred. For in vivo applications, useful exemplary final nucleic acid concentrations can be up to 5 mg/mL.

The composition of the present invention can be stored in aqueous suspension or can be dried. Therefore, in a preferred embodiment, the composition of the present invention is stored in dry, optionally freeze-dried (lyophilized) form. In a more preferred embodiment, the dried or lyophilized complex or composition also comprises a lyoprotectant. Lyoprotectants are molecules that protect (freeze-dried) material. Such molecules are normally polyhydroxy compounds such as sugars (mono-, di- and polysaccharides), polyalcohols and their derivatives. Trehalose and sucrose are known to be natural protectants for drying processes. Trehalose is produced by a variety of plants, fungi, and invertebrate animals that remain in a state of suspended animation during periods of drought (also known as anhydrobiosis). Sugars such as trehalose, lactose, raffinose, sucrose, mannose, sorbitol, mannitol, xylitol, polyethylene glycol, dextrins, urea, maltodextrins, fructans, maltooligosaccharides, manno-oligosaccharides, cycloinulohexaose, hydroxyethyl starch, dextrans, inulin, polyvinylpyrrolidone or amino acids such as tryptophan, Glycine and phenylalanine are particularly suitable lyoprotectants within the scope of the present invention. Most preferably trehalose is used in this context.

Pharmaceutical aspects

The composition of the present invention or the copolymer of the present invention can be used to deliver a nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, to target tissue or into a target cell. The term "delivering a nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, to a cell" preferably means the transfer of the nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, into the cell. Such use may be in vivo or in vitro.

The compositions of the present invention may also be used in a method of delivering a nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, to a target cell or tissue comprising the step of contacting a composition according to the invention with the target cell or tissue. Such a method can be carried out in vitro or in vivo. The in vivo method is not claimed. Contacting can be achieved by means and methods known to those skilled in the art. For example, if the method is carried out in vitro, contacting can be achieved by culturing the cells in the presence of the composition in the culture medium or by adding the composition to the cells. If the method is carried out in vivo, contacting with cells or tissues can be achieved, for example, by administering the composition to an individual by routes of administration. known to those skilled in the art, in particular by any route of administration that is commonly used in the field of gene therapy. Possible ways of formulating the composition and administering it to an individual are also further described below.

The term "in vivo" refers to any application to the body of a living organism wherein said organism is preferably multicellular, more preferably a mammal, and most preferably a human. The term "in vitro" refers to any application to body parts of a living organism isolated from and outside of said organism, eg, cells, tissues, and organs, wherein said organism is preferably multicellular, more preferably a mammal, and most preferably a human.

The present invention also relates to a pharmaceutical composition comprising the composition according to the invention and optionally a pharmaceutically acceptable carrier and/or diluent. In this context, it will be understood that the composition according to the invention as defined herein may be/can be used as a pharmaceutical composition on its own. The term "pharmaceutical composition" refers to a pharmaceutically acceptable form of the composition of the present invention that can be administered to a subject. The pharmaceutical composition is suitable for use in the treatment of the human or animal body by therapy (including prophylaxis).

The term "pharmaceutically acceptable form" means that the composition is formulated as a pharmaceutical composition, wherein said pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and/or diluent. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, or sterile solutions. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any subject depend on many factors, including subject size, body surface area, age, the particular compound to be administered, gender, time, and route of administration. of administration, general health and other drugs that are administered simultaneously. A typical dose of active substances may be, for example, in the range of 1 ng to several grams. Applied to nucleic acid therapy, in particular RNA, preferably single-stranded RNA such as mRNA, the dosage of the nucleic acid for its expression or for the inhibition of expression should correspond to this range; however, doses below or above this range are envisioned by way of example, especially considering the factors mentioned above. In general, the regimen as regular administration of the pharmaceutical composition should be in the range of 0.1 µg to 10 mg units per kilogram of body weight per day. If the regimen is a continuous infusion, it should also be in the range of 1 µg to 10 mg units per kilogram of body weight, respectively. Progress can be monitored through periodic evaluation. Dosages will vary but a preferred dosage for intravenous administration of nucleic acid, in particular, RNA, preferably single-stranded RNA such as mRNA, as constituents of the composition of the present invention, is about 106 to 1019 copies of the nucleic acid molecule. .

The term "administered" encompasses any suitable method of introducing the composition into the body of a subject. Administration of suitable compositions can be accomplished in a variety of ways, for example, by intravenous, intraarterial, intraperitoneal, subcutaneous, transdermal, intrathecal, intramuscular, topical, intradermal, intranasal, pulmonary inhalation, or intrabronchial or oral or rectal administration. The compositions of the present invention may be administered in particular as a gene-activated matrix as described by Shea et al. (Shea et al. 1999, Nat Biotechnol, 17, 551-554) and in EP1198489.

In principle, the pharmaceutical compositions of the invention can be administered locally or systemically. Administration will preferably be parenteral, for example intravenous, although other forms of administration are within the scope of the invention. Administration directly to the target site, eg, by catheter to a site in a blood vessel, is also conceivable. Administration can, for example, also occur by direct injection into a target site such as a tumor. Also within the scope of the invention is administration by aerosolization or nebulization or oral administration. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, fluorocarbons, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, and electrolyte replenishers (such as those based on Ringer's dextrose). Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases may also be present. Furthermore, the pharmaceutical composition may comprise additional agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.

A pharmaceutical composition according to the present invention can be used in a method of treatment (not claimed) comprising administering the pharmaceutical composition of the present invention to a patient for causing the nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, contained in said composition, to cause a preventive or therapeutic effect. Notably, the term "patient" encompasses animals and humans.

By administering the pharmaceutical composition of the present invention, diseases can be treated or prevented. The term "disease" refers to any conceivable pathological condition that can be treated, prevented or vaccinated against using an embodiment of the present invention. In a preferred embodiment of said method, said diseases may be hereditary, acquired, infectious or non-infectious, age-related, cardiovascular, metabolic, intestinal, neoplastic (in particular cancer) or genetic. A disease can be based, for example, on irregularities of physiological processes, molecular processes, biochemical reactions within an organism which in turn can be based, for example, on the genetic equipment of an organism, on behaviour, social or environmental factors such as such as exposure to chemicals or radiation. In a particularly preferred embodiment, the pharmaceutical composition of the present invention is used for treatments as disclosed in patent application WO2011/012316.

In line with the treatment method described above, the present invention relates in another embodiment to the use of the composition of the present invention for the preparation of a pharmaceutical composition for the treatment of a disease that can be treated by providing said nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, contained in said composition, to a tissue or organ within the body of a patient affected by a disease.

examples

Example 1

Materials:

Production of chemically modified Luc mRNA

To generate a template for in vitro transcription (IVT), plasmid pVAXA120-Luc was linearized by restriction digestion with NotI. The template was further purified by chloroform-ethanol precipitation. Template quality was determined by native agarose gel electrophoresis. IVT was performed with a standard IVT mixture containing ribonucleotide triphosphates and T7 RNA polymerase. Modifications were introduced using 25% 5-methyl-cytidine-5'-triphosphate and 25% 2-thio-uridine-5'-triphosphate. Capping was performed using the vaccinia virus capping enzyme, rGTP, and S-adenosylmethionine (SAM) as a methyl donor to add a 7-methylguanylate (m7GpppG) 0-cap structure to the 5' end of the mRNA. Purification of mRNA was performed by ammonium acetate precipitation. The modified Luc RNA was resuspended in water for injection and quality control was performed using UV measurement, native agarose gel electrophoresis, and transfection into NIH3T3 cells.

Polymers and polymer repeat unit average molecular weights:

Polymers were synthesized from aziridine (E), azetidine (P) or a stoichiometric mixture of aziridine (E) and azetidine (P) at monomer molar ratios (E:P) of 1:0, 0.8:1 and 0:1. Aziridine and azetidine were homopolymerized and copolymerized starting from a solution of the monomers in water (total concentration of monomer(s): 50% w/w). Polymerizations were carried out at 130 °C for 20-70 h with 0.001 equivalents of sulfuric acid. Homopolymers with an E:P ratio of 1:0 and 0:1, respectively, were produced for comparison purposes.

In this way, random branched copolymers of repeating units of ethylamine (-CH ² -CH ² -N<;“E”; molecular weight 42 g/mol) and propylamine (-CH ² -CH ² -CH ² - N<;"P"; molecular weight 56 g/mol) reflecting the stoichiometry of the monomers. The average molecular weight of the polymerized units was calculated according to the formula

This produces the following results:

Stock solutions:

Polymer stock solution: 0.5 mg/mL in water for injection

mRNA stock solution: 0.05 mg/mL stock solution in water for injection

N/P ratio and preparation of complexes of mRNA with polymers

The N/P ratio reflects the molar ratio of nitrogen input in a given amount of polymer to phosphate in a given amount of mRNA used to prepare a polymer-mRNA complex. The average molecular weight of a ribonucleotide monophosphate in an mRNA is 346 g/mol.

Therefore, the volume of polymer stock solution (vpolymer) of a given concentration (cpolymer) required for a given weight of mRNA (warn) at a desired N/P ratio is as follows:

Therefore, the volumes of polymer stock solutions given in the table below were diluted with the given volumes of water for injection to 25 µl in individual wells in rows A and E, respectively, of a 96-well plate.

Subsequently, 25 µl of mRNA stock (corresponding to 1.25 µg of mRNA) was added to the polymer solutions to produce the desired N/P ratio. Wells in rows B to D and F to H, respectively, were provided with 25 µl of water for injection each. After 30 min incubation, 25 µl of the complexes from row A or E, respectively, were transferred to row B or F, respectively using a multichannel pipette and mixed. Subsequently, 25 µl were transferred from row B to row C or from F to G, respectively, mixed and from C to D or G to H, respectively.

Cells, transfection and luciferase assay

HEK293 cells (DSMZ ACC305, Lot21) were grown in DMEM, MEM, MEM each including 10% FBS 1% P/S. Twenty-four hours prior to transfection, cells were seeded at a density of 5,000 cells in 100 µl of cell culture medium in a 96-well plate. Twenty µl of each of the polymer-mRNA complex dilution series were transferred to cells, corresponding to 125/62.5 ng of mRNA per well. After 24 h of incubation, cell culture supernatants were removed, cells washed with PBS, and then provided with 100 µl lysis buffer (25 mM Tris HCl, 0.1% TritonX 100, pH 7, 8).

Luciferase assay: 80 µl of the lysate was loaded into one well of a white 96-well plate and used for measurement of luciferase activity on a Wallac Victor2 instrument (Perkin Elmer). For this purpose, 100 µl of luciferase assay reagent (0.5 mM D-luciferin, 0.3 mM coenzyme A, 33 mM DTT, 0.5 mM ATP, 1 mM magnesium carbonate, 0.5 mM magnesium sulfate) was added. 2.7 mM, 0.1 mM EDTA, 20 mM tricine) and chemiluminescence was determined. The experiments were performed in triplicate.

Results:

The experiment shows that chemically modified Luc mRNA complexed with copolymer (0.8/1) is expressed more efficiently after cell transfection than complexed with either branched PEI (1/0) or PPI (0). /1) (figure 1). Taken together, this shows that the object of the present invention can be adequately addressed by the method and pharmaceutical preparations according to the present invention.

Example 2

In vivo aerosol application of chemically modified mRNA encoding firefly luciferase (Luc) formulated with copolymers to pig lungs

Chemical products

See example 1 above

Production of chemically modified Luc mRNA

See example 1 above

Experimental procedure

Pig sedation was initiated by premedication with azaperone 2 mg/kg body weight, ketamine 15 mg/kg body weight, atropine 0.1 mg/kg body weight, and followed by insertion of an intravenous line into the vein. side earphone. The pig was anesthetized by intravenous injection of propofol 3-5 mg/kg body weight as required. Anesthesia was maintained with continuous intravenous infusion of 1% propofol as required. Ventilation parameters were matched to end-expiratory carbon dioxide and adjusted if necessary. Anesthesia, respiratory and cardiovascular parameters were continuously monitored using pulse oximetry, capnography, rectal temperature probe, and reflex status. The pig received balanced electrolyte solution infusion at 10 ml/kg/h. The duration of anesthesia was approximately 80-120 min. The pig was euthanized with bolus injection of pentobarbital 100 mg/kg body weight through the lateral ear vein after completion of aerosol application (Aeroneb mesh nebulizer). Lungs were excised and approximately 1 cm thick tissue samples were cut from various lung regions followed by incubation in cell culture medium for 24 hours at 37°C (5% carbon dioxide) in an incubator. For measurement of luciferase activity, tissue samples were incubated in a bath of medium comprising D-luciferin substrate in PBS (100 µg/ml) at 37 °C for 30 min and subjected to bioluminescent imaging. ex vivo luciferase assay (IVIS 100, Xenogen, Alameda, USA).

Preparation of copolymer-mRNA polyplexes

Polyplexes were formed using a two-channel syringe pump (KDS-210-CE, KD Scientific). mRNA and copolymer (0.8/1, 1/0, 0/1 or 25 kDa PEI) each were diluted in 12.0 ml double distilled water resulting in a concentration of 500 μg/ml mRNA ( concentration of 25 kDa branched copolymer or PEI corresponding to an N/P ratio of 10). Both solutions were loaded into a separate 20 ml syringe using the draw function of the syringe pump at a rate of 5 ml/min. To mix both samples, the two syringes were connected via tubing (Safeflox Extension Set, B. Braun) to a t-piece. Mixing was done using the infusion function of the syringe pump at a rate of 40 ml/min. The complexes were incubated for 30 minutes at room temperature before use. It was observed in the course of the present invention that it is specifically advantageous to use only water without any buffer for complex formation because the nanoparticles may otherwise aggregate or be ineffective in the lungs of mice (Rudolph et al., J Mol Ther 2005, 12: 493-501).

Results:

Experiment shows that chemically modified Luc mRNA complexed with copolymer (0.8/1) is more efficiently expressed in lung cells of a pig after lung aerosol administration than complexed with 25kDa PEI (1/0). or PPI (0/1) (figure 2). Taken together, this shows that the object of the present invention can be adequately addressed by the method and pharmaceutical preparations according to the present invention.

Example 3

In vivo application of chemically modified mRNA encoding firefly luciferase (Luc) formulated with copolymers to the lungs of mice

Chemical products

See example 1 above

Production of chemically modified Luc mRNA

See example 1 above

Experimental procedure

Polyplexes of a 0.8/1 branched copolymer (ethylamine/propylamine units) ("br-Homo") and mRNA encoding firefly luciferase, and of branched polyethyleneimine ("br-PEI") and the mRNA were prepared and were applied either as an intratracheal fluid (n = 3), (25 µg of mRNA in a 100 µl volume) or as an intratracheal spray (12.5 µg of mRNA in a 50 µl volume) using a high-pressure Microsprayer device (PennCentury, USA). All experiments were approved by the local authorities (Regierung von Oberbayern) and were performed in accordance with the German animal welfare law. Adult female Balb/c mice were anesthetized in an isoflurane inhalation chamber. Subsequently, the test formulations were applied directly to the trachea using a custom light source and a small animal spatula. Animals recovered from anesthesia within minutes. After 6 hours, the animals were anesthetized by intraperitoneal injection of fentanyl/midazolam/medetomidine (0.05/5.0/0.5 mg/kg body weight). D-luciferin (1.5 mg diluted in 50 µl PBS) was applied to the nostrils of anesthetized animals and thus inhaled into the deeper airways. Bioluminescence imaging was performed using an IVIS Lumina XR imaging system (Perkin Elmer, USA). The results are shown in figure 3.

Description of the figures

Figure 1

Polyplexes were formed using 0.8/1, 1/0 or 0/1 copolymer (ethylamine/propylamine units) and mRNA encoding firefly luciferase at the indicated N/P ratios. After 24 h, HEK293 cells transfected with different amounts of mRNA (125 or 62.5 ng) were lysed and analyzed for luciferase activity.

Figure 2

Polyplexes were formed using 0.8/1, 1/0, or 0/1 copolymer (ethylamine/propylamine units), PEI (and commercially available branched PEI, 25 kDa, Sigma-Aldrich), and mRNA encoding firefly luciferase at a N/P ratio of 10. Twenty-four hours after aerosol administration in pigs, the lungs were removed and analyzed for firefly luciferase activity by bioluminescent imaging of lung tissue sections.

Figure 3

Polyplexes of a 0.8/1 branched copolymer (ethylamine/propylamine units) ("br-Homo") and mRNA encoding firefly luciferase, and of branched polyethyleneimine ("br-PEI") and the mRNA were applied to mice. as an intratracheal liquid and as an intratracheal spray. The figure shows the results of bioluminescence imaging tests using an IVIS Lumina XR imaging system (Perkin Elmer, USA).

Claims (15)

i. A statistical copolymer comprising a plurality of repeating units (a) independently selected from repeating units of the following formulas (a1) and (a2):

, Y a plurality of repeating units (b) independently selected from repeating units of the following formulas (b1) to (b4):

wherein the molar ratio of the sum of repeat units (a) to the sum of repeat units (b) is within the range of 0.7/1.0 to 1.0/0, 7, and wherein one or more of the nitrogen atoms of the repeating units (a) and/or (b) contained in the copolymer may be protonated to provide a cationic copolymer. 2. The copolymer according to claim 1, which is a branched or dendritic copolymer comprising one or more types of repeating units selected from repeating units (a2), (b2) and (b4). 3. The copolymer according to claim 1, which is a linear copolymer comprising repeating units (a i) and (b1). 4. The copolymer according to any of claims 1 to 3, wherein the repeating units (a) and (b) represent 80 mol% or more of all the repeating units in the copolymer. 5. The copolymer according to any of claims 1 to 3, wherein repeating units (a) selected from (ai) and (a2) and repeating units (b) selected from (b1) and (b2) represent 80 mol% or more of all repeating units in the copolymer. 6. The copolymer according to any of claims 1 to 5, wherein the terminal groups of the copolymer comprise one or more types of groups (c) independently selected from groups of the formulas (c1) to (c3):

7. The copolymer according to any of claims 1 to 6, wherein the molar ratio of repeating units (a) to repeating units (b) is within the range of 0.8/1.0 at 1.0/0.8. 8. The copolymer according to any of claims 1 to 7, which can be obtained by polymerizing a mixture of monomers comprising aziridine, azetidine and optionally pyrrolidine. 9. A composition comprising a nucleic acid and a copolymer according to any one of claims 1 to 8. 10. The composition according to claim 9, wherein the nucleic acid is mRNA. The composition according to claim 9 or 10, wherein the copolymer is a cationic copolymer, and wherein the cationic copolymer forms a complex with the nucleic acid. 12. A pharmaceutical composition comprising a composition according to any one of claims 9 to 11, and optionally a further pharmaceutically acceptable diluent and/or carrier. 13. A copolymer according to any one of claims 1 to 8, or a composition or a pharmaceutical composition according to any one of claims 9 to 12 for use in therapy by delivering a nucleic acid to a cell. 14. An in vitro method for delivering a nucleic acid to a target cell or tissue comprising the step of contacting a composition or pharmaceutical composition according to any one of claims 9 to 12 with the target cell or tissue. 15. A method for the production of the copolymer according to any one of claims 1 to 7, comprising the step of polymerizing a mixture of monomers comprising aziridine, azetidine and optionally pyrrolidine.